Cellular inflammatory response of rainbow trout to PKX
by Elizabeth MacConnell
A thesis submitted in partial fullfillment of the requirements for the degree of Master of Science in
Veterinary Science
Montana State University
© Copyright by Elizabeth MacConnell (1988)
Abstract:
The cellular inflammatory response of rainbow trout to PKX was investigated. The period studied was
from three to twenty weeks post-injection of PKD-infected kidney homogenate. The inflammatory
response to PKX was compared to that of a non-infectious agent, bentonite clay. Kidney samples were
examined by light and electron microscopy.
Cell identification was based on the literature for peripheral blood leukocytes. In contrast to most
Myxosporeans, PKX provoked a severe host response. Initially, the response to PKX was hempoietic
hyperplasia followed by proliferation of mononuclear cells. The major lesion was a marked
granulomatous nephritis. Resolution of lesions without fibrosis and elimination of PKX was seen by
termination of the study. The macrophage was the predominant cell type involved in the inflammatory
response to PKX throughout the study. Clay induced a chronic granulomatous response only in the
viscera. Intense proliferation of melanomacrophages was the predominant response seen in kidneys,
but clay particles that reached the kidney were phagocytosed primarily by macrophages. In this study,
PKX and a non-infectious agent, clay, were effectively removed from the kidney by macrophages.
Hemtopoietic cell types in fish are still far from being adequately charcterized, in particular the
immature forms. CELLULAR INFLAMMATORY RESPONSE OF RAINBOW TROUT TO PKX
by
Elizabeth MacConnell
A thesis submitted in partial fulIfillment
of the requirements for the degree
of
Master of Science
in
Veterinary Science
MONTANA STATE UNIVERSITY
Bozeman, Montana
May 1988
M 133%
ii
APPROVAL
of a thesis submitted by
Elizabeth MacConnell
This thesis has been read by each member of the thesis
committee and has been found to be satisfactory regarding
content, English usage, format, citations, bibliographic
style, and consistency, and is ready for submission to the
College of Graduate Studies.
Approved for the Major Deparment
Approved for the College of Graduate Studies
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulIfilIment of
the requirement for a master's degree at Montana State
University, I agree that the Library shall make it
available to borrowers under rules of the Library.
Brief
quotations from this thesis are allowable without special
permission, provided that accurate acknowledgement of
source is made.
Permission for extensive quotation from or
reproduction of this thesis may be granted by my major
professor, or in his/her absence, by the Dean of Libraries
when, in the opinion of either, the proposed use of the
material is for scholarly purposes.
Any copying or use of
the material in this thesis for financial gain shall not be
allowed without my written permission,
Signature^
Date
iv
ACKNOWLEDGEMENTS
I would like to express my appreciation to my major
advisor Dr. C.A.
Speer for his support; Drs. D . Young and
D • Worley for their cooperation; my mentor Mr. C.E.
Smith
for his guidance, support and continued encouragement; Dr.
R • p * Hedrick for providing samples for the PKD study,
valuable suggestions and assistance; Mr. A.
Blixt for his
technical assistance in electron microscopy; Ms. G . Callis
for staining advice; Drs H . Ferguson and D . Hinton for
their interpretations of electron micrographs; and to my
family, especially Jamie and Randy, for their continued
support and encouragement.
V
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS..... ................... ............ . iv
LIST OF FIGURES....................
vii
ABSTRACT............. ....... ....................... . ...x
INTRODUCTION.....................................
i
LITERATURE REVIEW.................
4
Historical Background.............................. 4
Etiological Agent.................................. 4
Taxonomy....................
4
Morphology.............
5
Epizootiology....... ............................... .
Host and Geographical Location................ 8
Transmission.........
9
Environmental Factors...................... . .10
Pathogenesis. ..................................... ..
Control...............
15
Immunity...... ................................... .
Host Response to PKX..........
18
Host Response to Non-Infectious Material...... 20
MATERIALS AND METHODS........
22
Host Response to PKX.............
22
Animals .................
22
Experimental. Protocol.................
23
Tissue Collection and Processing ............ ,..23
Host Response to Clay......
25
Anima Is...............
.25
Experimental Protocol...................... ...25
Tissue Collection and Processing............. 26
Cell Classification.......
26
RESULTS..........................
30
PKX............................................... .
Host Response to PKX............................. 34
Week Three........
34
Week Five.......
40
Week Seven..........
....50
Week Ten..................................... 55
vi
Week Twenty..........
Host Response to Clay. ......
Week One....... ......
Week Three;..........
Week Five............
Week Seven...........
Week Ten..........
Week T w e n t y . .....
61
65
65
67
69.
70
73
79
DISCUSSION....... ...........
81
LITERATURE CITED
88
vii
LIST OF FIGURES
Figure
Page
I.'
PKX containing internal daughter cell
2.
PKX containing daughter cells that appear to be
separating from enveloping primary cell.......... . 33
3.
PKX in sinusoid with macrophage closely adhered
to cell surface, three weeks PI. PKX containing
daughter cell in kidney interstitium..... ........35
4.
Vacuoles containing cellular material in tubular
epithelium. Lymphocytes migrating through
tubular epithelium.............................
5.
Migrating lymphocyte and vacuoles in tubular
epithelium and granular material in tubular
lumina, three weeks PI...... ......... . .........i.38
6.
Circulating macrophage attached to endothelium
of vessel wall, three weeks PI......... ..........39
7.
Unidentified cell found in kidney interstitium,
three weeks PI.... ..................... .
8.
Unidentified cell with electron-dense cytoplasm,
vacuoles, idented nucleus and pseudopodia........ 42
9.
Mild PKD.
..32
Severe PKD, five weeks PI.............. 43
10.
PKX in tubular epithelium and .lumina. PKX in .
tubular epithelium and hemopoietic tissue,
five weeks PI..... ..........^
...... 45
11.
Vascular lesion in renal vein, five weeks PI.
Vascular lesion containing PKX, lymphocytes,
macrophages and erythrocytes.......... .......
46
Macrophages closely adhered to the cell
surface of PKX........... ............ .........
Aft
12.
viii
13.
PKX in kidney circulation partially and
completely engulfed by macrophages..... ..... ....49
14.
Macrophage containing degenerate cellular
material, possibly PKX, in tubular
epithelium, five weeks PI....... .................51
15.
Granulomatous lesions seven weeks PI.
PKX in the center of a whorl of macrophages....... 52
16.
Epithelioid tissue seven weeks. PI................ 54
17.
Macrophage adhered to surface of PKX in
kidney interstitium. Melanin-like material
lining tubular lumina ten weeks PI............... 56
18.
Macrophages closely adhered to the surface
of PKX..........
.58
19.
Electron-dense granules in close proximity
to plasmalemma adjacent to PKX.............. . .... 59
20.
PKX with macrophages closely adhered to cell
surface and lymphocyte closely associated
with macrophages......................
N
60
21.
Macrophage migrating through basal lamina
into tubular epithelium, ten weeks PI............. 62
22.
Thrombocytes found at ten weeks PI........
23.
Regenerating tubule, twenty weeks PI.............. 64
24.
Thickened basal lamina and regenerating
tubular epithelium, twenty weeks PI..............66
25.
Clay particles contained in macrophage which
is overlying endothelial cell..................... 68
26.
Melanomacrophages found in kidney interstitium....71
27.
Visceral granuloma seven weeks PI.
Melanomacrophages dominate the kidney
interstitium seven weeks PI............... ....... 72
63
ix
28.
Macrophage with phagocytosed clay particles
seven weeks PI.... ...................... ..... ..74
29.
Large, electron-dense absorption granules in
proximal tubular epithelium seven weeks PI........75
30.
Lymphocytes and fibroblasts in visceral
granuloma. Melanomacrophages in the kidney
circulation ten weeks PI...... ........... ...... 77
31.
Plasma cell adjacent to kidney,tubular
epithelium with small barbelI-shaped
mitochondria ten weeks PI. ................ ...... 78
32.
Melanocytes in visceral granuloma twenty
weeks PI.... ....................... ............ 80
X
ABSTRACT
The cellular inflammatory response of rainbow trout to
PKX was investigated. The period studied was from three to
twenty weeks post-injection of PKD-infected kidney
homogenate. The inflammatory response to PKX was compared
to that of a non-infectious agent> bentonite clay. Kidney
samples were examined by light and electron microscopy.
Cell identification was based oh the literature for
peripheral blood leukocytes. In contrast to most
Myxosporeansf PKX provoked a severe host response.
Initially, the response to PKX was hempoietic hyperplasia
followed by proliferation of mononuclear cells. The major
lesion was a marked granulomatous nephritis. Resolution of
lesions without fibrosis and elimination of PKX was seen by
termination of the study. The macrophage was the
predominant cell type involved in the inflammatory response
to PKX throughout the study. Clay induced a chronic
granulomatous response only in the viscera. Intense
proliferation of melanomacrophages was the predominant
response seen in kidneys, but clay particles that reached
the kidney were phagocytosed primarily by macrophages. In
this study, PKX and a non-infectious agent, clay, were
effectively removed from the kidney by macrophages.
Hemtopoietic cell types in fish are still far from being
adequately charcterized> in particular the immature forms.
I
INTRODUCTION
Proliferative kidney disease (PKD) is a potentialIy
severe parasitic disease of economic importance in
intensively cultured salmonid fishes in Europe and North
America.
This disease is caused by an unclassified
'
protozoan referred to as PKX. It has been proposed that PKX
belongs to the phylum Myxozoa because myxosporean
trophozoites and developing spores have been observed in
the renal tubules of PKD-infected fish (Hedrick et al.,
1984; Kent and Hedrick, 1985).
Identification of the
infective stage, mode of transmission, intermediate or
reservoir hosts and source of the infection are unknown..
In addition, the life cycle of this parasite is poorly
understood.
Proliferative kidney disease was initially described
in fingerling rainbow trout (Salmo qairdneri) by Roberts
and 'Shepherd in 1974.
Since then, PKD has been reported in
most European countries (Clifton-Hadley et al., 1984).
The
fi^st reported outbreak of PKD in North America occurred in
rainbow trout in 1981, at the Hagerman State Fish Hatchery,
Hagerman, Idaho (Smith et al., 1984).
2
Typically, PKD affects fingerling trout or salmon of ■
>
the 0+ year age class.
The disease has been reported in
several trout and salmon species, most often in rainbow
trout.
Outbreaks of PKD vary markedly in severity;
morbidity can be 100% with mortality from 0% to 90%
(Clifton-Hadley et al., 1984).
Fish with PKD show poor
tolerance to stress, increased feed conversion rate and
greater susceptibility to secondary infections.
It has been suggested that salmonid species are
aberrant hosts for this parasite due to incomplete spore
development and the severe inflammation seen in infected
fish, which is unusual for most myxosporean infections
(Dykova and Lom, 1978).
Similar characteristics shared
between the family Shaerosporidae and PKX have been
described by Hedrick et al.(1984; 1988) and Kent (1985).
Shaerospora sp. has been found in tui chub (Gila bicolor)
inhabiting the water supply of PKD-infected Hot Creek State
Hatchery, California and stickleback (Gasterosteus
aculeatus) from Quinault Lake, Washington, water supply of
a PKD-infected steelhead hatchery (Hedrick et al., 1988).
Although Shaerospora sp. has been implicated as the
possible etiologic agent of PKD, cross transmission
experiments conducted by Rafferty (1985) with Shaerospora
infected roach (Rutilus rutilus) and carp (Cyprinus
3
carpio), and by Hedrick (pers.
comm.) with tui chub and
stickleback have been unsuccessful.
The initial cellular response observed by light
microscopy to PKX is the proliferation of hemopoietic cells
in the interstitium of the kidney.
Subsequently., the major
kidney tissue response to PKX is interstitial
hypercellularity, attributed to the infiltration and
proliferation of mononuclear cells.
There are variable
reports in the literature concerning the cells involved in
the immune response to PKX. A variety of morphological and
staining characteristics have been used to describe these
cells, but the cell types have not been defined.
The
objective of this study was to describe, by light and
electron microscopic examination of rainbow trout kidney,
the predominant cell types involved in the inflammatory
response to a non-infectious agent and to PKX during
initial and later stages of disease.
I
4
LITERATURE REVIEW
Historical Background
A disease syndrome in fingerling rainbow trout with
gross -kidney changes was first named prolferative kidney
disease (PKD) by Roberts and Shepherd in 1974.
Subsequently, Ferguson and Needham presented the first
indepth description of PKD (1978).
Possibly, earlier
diseases described as "amoebiasis" of trout (Plehn, 1924),
infectious kidney swelling and liver degeneration
(Schaperclaus, 1954) and infectious anemia (Besse, 1954)
were actually PKD. Proliferative kidney disease is now
considered one of the most devastating diseases in the fish
farming industry in Europe.
Etiological Agent
Taxonomy
Initially, PKD was thought to be caused by an amoeba
because the organism formed pseudopodia (Ghittino et al.,
1977).
Ghittino et al.
(1980) subsequently concluded that
this amoeba was a contaminant.
Ferguson and Adair (1977)
suggested that PKX was an amoeba or myxosporean but the
5
inability to culture the organism or find spores precluded
a definite classification.
An ultrastructural study of the
PKX parasite conducted by Seagrave et al.
(1980) showed
haplosporean features, namely the occurrence of what they
referred to as
haplosporosomes", and multivesicular bodies
in the cytoplasm; similar to those seen in the oyster
parasite, Martiela sp., currently in the phylum Acetospora.
Structures similar to "haplosporosomes" have also been
described from members of the phylum Myxozoa (Current,
.1979).
Recent studies by Kent and Hedrick (1985) indicate
that PKX is a myxosporean, as evidenced by the presence of.
intraluminal trophozoites and developing spores in kidney
tubules of infected steelhead trout.
The fine structural
features of PKX as described by Feist and Bucke (1987) are
consistent with early stages of sporogenesis of a
myxosporean.
However, the precise taxonomic status of PKX
has yet to be determined.
Morphology
PKX organisms are 5-20 urn in diameter, stain weakly
eosinophilic with H&E, have periodic acid-Schiffs (PAS)
positive cytoplasmic granules, small pseudppodia, distinct
plasmalemma and contain one to several secondary or
daughter cells (Ferguson and Needham, 1978; Hedrick et al.,
1984; Smith et al., 1984).
DltrastructuaIIy, a prominent
6
feature of PKX is the presence of electron-dense
cYtoplasinic inclusions, referred to as "haplosporosomes"•
(Seagrave et al., 1980).
They are 0.14-0.20 um in
diameter, have an electron-lucent bar, are often associated
with the plasmalemma and are found only in the primary cel]
(Ferguson and Needham, 1978).
The nature and function of
PKX "haplosporosomes" are not known.
The most consistently described characteristics of the
PKX primary cell are a well developed endoplasmic
reticulum, numerous multivesicular bodies, lipoid bodies
and a prominent plasmalemma (Kent and Hedrick, 1984).
Secondary or daughter cells found within primary cells have
a distinct Golgi apparatus, double cell membranes and
numerous cytoplasmic ribosomes (Feist and Bucke, 1987)
typical of myxosporean generative cells (Current 1979).
Bundles of microtubules within secondary and tertiary cells
have also been observed, an indicative feature of
myxosporeans (Feist & Bucke 1987).
Two spherical polar
capsules with coiled filaments found within capsulogenic
cells have been described in spores developing in kidney
tubules of steelhead and rainbow trout (Kent and Hedrick,
1985).
Many similarities exist between PKX and early stages
of a Shaerospora sp. first described as "Csaba's blood
(
protozoan", in the blood of common carp (Cyprinus carpio)
7
(Bucsek and Csaba, 1981).
Further studies determined that
the Csaba's blood protozoan was an early stage of S .
renicola and the parasite that causes swimbladder
inflammation (Molnar, 1984; Csaba et al., 1984).
Shaerospora renicola forms endogenous daughter cells which
are released and sporulate in kidney tubules, similar to
the pattern of development that has been observed for PKX
(Csaba et al., 1984; Kent and Hedrick, 1985).
Development of intraluminal forms of PKX are similar
to the sporogenic stages of Shaerospora spp. Both
myxosporeans are monosporous and sporoblasts are formed
within pseudoplasmodia.
The outer enveloping cell of the
intraluminal PKX is analogous to the pseudoplasmodia
described for Shaerospora (Lorn et al., 1983).
Although
there is no evidence of valve formation, valvogenic cells
surrounding PKX capsulogenic cells have been observed and
the immature spores of PKX and Shaerospora are very similar
in size and shape (Kent and Hedrick 19.85) .
Shaerospora has been reported in brown trout from PKD
enzootic waters (Ferguson, 1984; Fischer-Scherl et al.,
1986) and one rainbow trout from a hatchery where PKD is
enzootic (Hedrick et al., 1988).
8
Epizootioloqy
Host and Geographical Location
Proliferative kidney disease is found principally in
salmonid species, most commonly in hatchery-reared rainbow
trout.
The disease has also been reported in cultured
chinook salmon,(Oncorhynchus tshawytscha) and coho salmon
(O .
kisutch) (Hedrick et al,, 1984); Atlantic
salmon,(Salmo salar) and brown trout,(S »
trutta) (Ellis et
al., 1982); wild grayling, (Thyma11us thymalIus) (Seagrave
et al., 1981) arctic char, (Salvelinus alpinus) (Bucke et
al., 1985).
Northern pike, (Esox lucius) and roach,
(Rutilus rutilus) are the only non-salmonid species in
which PKD has been described (Seagrave et al., 1981).
Since proliferative kidney disease was first described
in 1974, it has been diagnosed in England, Wales, Scotland,
Northern Ireland, Sweden, Republic of Ireland, Italy,
France, Germany (Clifton-Hadley et al,, 1984) and Denmark
(Olesen et al., 1983).
Following the discovery of PKD in
1981 in Idaho (Smith et al., 1984), the disease has been
reported in hatcheries in California, Washington and
British Columbia, Canada (Hedrick et al., 1984; Hoskins,
1986).
This may not be indicative of an increased range of
the disease but improved recognition of PKD. Review of
9
histological records at the American River California State
Fish Hatchery since 1966 revealed the presence of PKD,
previously referred to as "lupus" (Hedrick et al., 1985).
Transmission
Natural transmission of PKX occurs by exposure to
enzootic waters.
There is no evidence of transmission from
fish to fish or by feeding homogenized or trypsinized
preparations of infected kidneys to healthy fish (Ferguson
and Ball, 1979, D'Silva et al., 1984).
Holding
disease-free fish in aquaria with feces from infected fish,
or with infected fish was also unsuccessful in transmitting
PKX (D 'Silva et al., 1984).
Experimental transmission of
PKX has been successful only by intraperitoneaI injection
of infected kidney homogenates (Clifton-Hadley et al.,
1984; D 'Silva et al., 1984), or with whole blood or spleen
homogenates (Kent et al., 1985).
There is little evidence as to the mode of entry of
the PKX parasite into the fish.
Access through the gill
(Clifton-Hadley et al., 1983) or ingestion (Ghittino et
al., 1977) have been suggested as the most likely, because
the parasite would then be able to travel via the lymphatic
and/or circulatory systems to the target tissues.
10
Environmental Factors
The severity of PKD can vary markedly and is probably
influenced by several environmental factors.
Low oxygen
levels and fish handling result in increased mortalities
due to the respiratory distress caused by the anemia
induced in infected fish.
The disease occurs most frequently at water
temperatures of 15 C or above (Clifton-Hadley et al.,
1984) .
Spontaneous infections and experimentally-induced
disease have occurred at lower water temperatures (Ellis et
al., 1982; Hedrick et al., 1984).
Several studies suggest
that onset and severity of the disease are dependent on
water temperature (Ferguson and Ball, 1979; Ferguson, 1981;
Clifton-Hadley et al., 1985; Foott et al., 1986).
Most
likely water temperature affects both the development of
the parasite and the host response (Clifton-Hadley et al.,
1986).
Time of peak infectivity may be seasonal due to the
cyclical development of the parasite, which may not be
associated with rising water temperature (Hedrick et al.,
1985) .
At one study site in California, the infective
stage of PKX was present in the water from April through
November with peak prevalence of infection occurring in
June (Foott et al., 1986).
Data from the Hagerman Idaho
State Fish Hatchery also support the seasonality of PKD. At
I
11
this facility the water temperature is a constant 15 G
throughout the year but PKD only occurs from April through
December (Smith, pers.
comm.)
Concurrent infections with bacteria, viruses and other
protozoans often occur, causing greater losses and making
cause of mortality more difficult to determine.
Initially,
PKD was reported to occur only in soft water conditions
(Roberts, 1978; Ferguson and Needham, 1978), but the
disease occurs in hard alkaline waters as well (Scott,
1979).
Pathogenesis
Initial diagnosis of PKD is made by microscopic
examination of imprint (Klontz and Chacko, 1983;
Clifton-Hadley et al., 1984) or wet mounts of kidney
prepared from affected fish.
Confirmation is obtained by
observation of the parasite and associated host
inflammatory response by histologic examination of affected
kidney tissue (Hedrick et al., 1984).
Serological
diagnostic tests are not available for the diagnosis of
PKD.
,
Six to eight weeks after infection, clinical signs
including anemia, abdominal distention due to ascites,
darkened coloration, exophthalmia, renalmegaly and
12
splenomegaly appear (Ferguson and Needham, 1978).
Presumptive diagnosis is often difficult because these
signs are often encountered with other fish diseases that
affect kidney function.
Affected kidneys are usually grey
and markedly swollen; in severe cases they appear
corrugated.
often anemic.
Fish exhibiting clinical signs of PKD are
The anemia has been classified as a chronic
hemolytic anemia, possibly caused by toxins released from
the parasite (Hoffman and LommeJ, 1984).
Leukocytosis has
not been reported in fish with PKD.
Gross or microscopic signs of PKD are rarely detected
before four weeks following natural or experimental
infection to PKX. The characteristic kidney lesions are
areas of diffuse granulomatous reaction that often surround
one or more PKX parasites (Hedrick et al., 1986).
Ferguson
and Needham (1978) described a whorling appearance of
inflammatory tissue with centrally placed PKX cells,
nephron destruction and sclerosis of glomeruli.
Severe
necrotizing vasculitis and subsequent occlusion of renal
and hepatic vessels are observed frequently due to PKX
organisms adhering to and destroying vessel walls (Smith et
al., 1984).
PKX can invoke a similar granulomatous
response in the spleen, gills, muscle, pancreas and a
mononuclear infiltrate in liver and intestinal mucosa of
heavily infected fish (Ferguson and Needham, 1978;
13
Clifton-Hadley et al., 1984).
The location of the parasite during early infection
is unknown.
Using antiserum to PKX from rabbits, Rafferty
(1986) was able to produce marked kidney tubular
fluorescence by the indirect immunofluorescent test (IFT)
during the one to three week period post-injection.
The
parasite was not observed in these samples during this
period.
Tubular fluorescence did not occur in control fish
and to a very small degree during the subsequent course of
the disease, but fluorescing PKX cells were observed in the
hemopoietic tissue from four to nine weeks post-injection.
Kent, and Hedrick (1986) observed an early form of PKX
in the kidney interstitium at three weeks post-exposure.
They described the organism as small, condensed,
eosinophilic, and containing a daughter cell.
Clifton-Hadley et al.
(1983) found the first evidence of
PKX at five weeks post-exposure in the peritubluar
capillaries, often with one or more basophilic,
crescent—shaped bodies associated with their outer surface.
PKX has been detected in tubular epithelium and lumina as
early as seven weeks after injection (Kent and Hedrick,
1986).
The intraluminal parasites further develop to form
multicellular spores with polar capsules, but no valves
(Kent and Hedrick, 1986).
The sequential pathologic changes described in the
14
literature for PKD varies in the time course from initial
response and lesion development to resolution.
The initial
cellular response to PKX, observed by light microscopy five
to seven weeks post-injection, is the proliferation of
hemopoietic cells in kidney tissue (Clifton-Hadley et al.,
1984).
Proliferation of melanomacrophage centers as a
feature of early lesions has also been reported (Rafferty,
1986).
The major kidney tissue response to PKX is
interstitial hyperceIlularity which is attributed to the
infiltration and proliferation of mononuclear cells,
presumably macrophages, and is prominent between eight to
eleven weeks post-injection (Kent and Hedrick, 1986).
These cells obliterate much of the normal hemopoietic
tissue, renal tubules and glomeruli by nine weeks
post-injection (Ellis et al., 1985).
The principal cell
types in lesions have been described tentatively as
macrophages, lymphocytes, fibroblasts and cells that may be
transforming into plasma cells (Ferguson and Needham, 1978;
Clifton-Hadley et al., 1983).
Lymphocytes are also
abundant and have been reported closely associated with PKX
(Clifton-Hadley et al., 1983; Kent and Hedrick, 1986).
Organization and resolution of kidney lesions and necrosis
of PKX has been reported by 12 weeks after onset of disease
(Ellis et al., 1982).
Nodules of chronic inflammation
containing intact PKX and fibrous tissue have also been
15
observed (Clifton-Hadley et al., 1983).
Resolution of
lesions involves destruction of the parasite and resorption
of dead and dying cells.
In most fish surviving PKD there
is little or no histologic evidence of lesions at 21 weeks
post-injection (Clifton-Hadley et al., 1983).
Control
Until recently the use of antibacterial and/or
antiprotozoal compounds had been unsuccessful in
controlling PKD (Ghittino et al;, 1977; Ferguson and Ball,
1979; Bucke et al., 1981; Clifton-Hadley et al., 1984).
Most recently, successful treatments of Shaerospora
renicola infections of common carp by oral administrations
of fumagillin, an antibiotic produced by the fungus
Aspergillus fumigatus, led Hedrick et al.
(1988) to test
the drug against PKD in juvenile chinook salmon.
The drug
provided protection against PKD but toxicity problems were
encountered.
Development of PKD was found to be delayed
when clinically and subclinicalIy affected fish were treated
with repeated doses of high concentrations of malachite
green (Clifton-Hadley and Alderman, 1987).
However,
unacceptable levels of malachite green were found in the
fish.
Further study on toxic effects of these chemicals on
fish will be necessary before they can be used for the
16
treatment of PKD in hatcheries.
In Scotland, salination of the water supply has been
reported to alleviate signs of PKD (O'Hara, 1985).
However, an experiment conducted in California in which
Chinook salmon with PKD were transferred to full strength
sea water did not demonstrate the same beneficial effects
(Hedrick and Aronstein, 1987).
Usually, modifications of
husbandry practices such as delayed movements to infected
water, reduced handling, lowered densities, increased
oxygen levels and decreased water temperatures are employed
to reduce the affects of PKD, increasing the economic cost
of the disease.
Immunity
Fish have non-specific, natural immunity and specific
humoral and celI-mediated immune mechanisms similar to, but
not identical with, those of higher vertebrates (Ellis and
Munroe, 1976).
A characteristic feature of both
celI-mediated and antibody-mediated immune response in fish
is their dependence on water temperature.
Several studies
on the relationship between water temperature and the rate
of development of the acute and early chronic inflammatory
response have shown an approximate 50% reduction in rate of
development for a reduction in temperature of 10 C (Finn
17
and Nielson, 1971; McQueen et al., 1973; Roberts et al.,
1973,; Anderson and Roberts, 1975).
There are three major lymphoid organs in fish: the
thymus, spleen, and kidney.
The anterior portion of the
kidney is the primary site of hemopoiesis.
Posteriorly,
much of the extrarenal tissue is also composed of
blood-forming tissue (Yasutake and Wales, 1983).
Many
undifferentiated cells, as well as immature and mature red
blood cells and white blood cells are present.
Based on
mammalian morphological criteria, fish leukocytes have been
classified into five main groupings: thrombocytes,
lymphocytes, granulocytes, monocytes and hemocytoblasts
(Ellis, 1977).
There is no clear division of lymphocytes
into T- and B-cells, although there is some evidence of
functional analogues to T- and B-cells (Lewis et al.,
1979).
Fish possess only one, Ig-M like, tetrameric
immunoglobulin class (Dorson, 1981).
Many features of the fish defense system are poorly
understood such as the role of the neutrophil, the function
of the complement system, the presence, nature and role of
histaminogenic cells and the nature, origin and possible
role of the melanin and lipofucsin pigments associated with
inflammatory lesions.
Morphologically the cells involved in a granulomatous
response are generally similar to their counterpart cells
18
in higher animals, both at the light microscopic and
ultrastructural levels (Timur and Roberts, 1977).
However,
the inflammatory response in fish is less intense and
slower to appear and resolve than in mammals (Finn and
Nielson, 1971).
Host Response to PKX
There are limited reports in the literature concerning
immunity to PKD. Yearlings not previously exposed to PKD
readily contract the disease, whereas survivors show
complete resistance to reinfection (Kent and Hedrick,
1985).
However, previous exposure alone is not sufficient
to produce immunity; recovery from active infection is
necessary (Ferguson and Ball, 1979; Kent and Hedrick,
1985).
The nature of resistance to infection most likely
results from strong humoral (Olesen and Jorgensen, 1985;
Klontz et al., 1985) and cellular responses (Ellis et al.,
1982).
Passive transfer of serum from recovering fish to
actively infected fish speeds the recovery and reduces the
incidence of parasites and lesions (Hedrick et al.,
1985).
Hypoproteinemia is also a characteristic of PKD. Scott
(1984) measured changes in serum protein levels during an
outbreak of PKD and suggested that the increase of one
protein, most likely an acute phase protein, was indicative
19
of s defense response•
Klontz et aI.
(1986) reported a
progressive increase in the serum beta globulins in the IgM
range for salmonids and decrease in albumin in clinically
ill fish.
D:iff®.rent stocks of Atlantic salmon have exhibited
diverse susceptibilities to infection with PKX (Ellis et
al•/ 1982).
Reports have also suggested different levels
of susceptibility to PKD based on the varied intensities of
the host response.
For example, Kent and Hedrick (1985)
observed a higher incidence of intraluminal parasites and
corresponding milder proliferative response in PKD-infected
kidneys of brown trout when compared to rainbow trout.
There are variable reports in the literature
concerning the cells involved in the immune response to
PKX. Giant cells, typical of Type IV delayed
hypersensitivity, have occasionally been described in brown
trout and Atlantic salmon but never in rainbow trout with
PKD (Ellis.et al., 1985).
Cells which may be transforming
into plasma cells have also been described (Ellis et'al.,
1985), however there is incomplete evidence for plasma
cells in fish.
Ferguson (1976) proposed that
immunoglobulin is produced by stimulated lymphocytes that .
do not fully differentiate into plasma cells.
Lymphocytes
found closely associated with PKX suggest an active immune
response (Olesen and Jorgenson, 1985).
20
Host cells associated with PKX are often described by
a variety of morphological and staining characteristics
such as: large frothy cells; small basophilic cells;
epithelioid in form (Ellis et al., 1985); basophilic,
crescent-shaped bodies; large sac-like cells
^dift-0IV-Hadley et al., 1984) ; and aberrant macrophages
(Rafferty et al., 1985).
Host Reponse to Non-Infectious Material
Carrageenin, a seaweed extract, has been used
successfully to induce granuloma in plaice (Pleuronecctes
Rlatessa), maintained in water at 10 C. Within 24 hours of
injection, a local inflammatory resonse consisting of
neutrophils, macrophages and lymphocytes, had developed
(Timur and Roberts, 1977).
There was no evidence of
intracellular carrageenin at this time.
'
Initial
phagocytosis was hot observed until day five and was not
completed until day 42 (Timur and Roberts, 1977).
By the
IOth day, the inflammatory cells were almost exclusively
macrophages.
Granulation tissue was still present at the
termination of the experiment on day 80 (Timur and Roberts,
1977).
Fish held in 10 C water showed all the features of
a marked granulomatous response by day 18, including
epithelioid development.
However, the first appearance of
macrophages with a distinctive foamy cytoplasm and
21
fibroblastic activity was not observed until day 18 in fish
held at 5 C (Timur and Timur, 1985).
Studies involving the intraperitoneaI (IP) injection
of colloidal carbon in plaice held in 8-10 C water have
shown that the carbon gains access to the circulation and
is phagocytosed primarily by the ellipsoids of the spleen
and the reticuloendothelial cells in the kidney and heart
(Ellis and Munroe, 1976).
These reticuloendothelial cells
became free macrophages and were highly phagocytic.
Aggregates of carbon containing macrophages in the kidney
and spleen were first observed within or on the periphery
of melandmacrophage centers, four days after injection
(Ellis and Munroe, 1976).
Ferguson (1984) found that
bacteria were phagocytosed by macrophages closely
associated with endothelium of the renal portal
circulation, but not by the endothelial cells.
/ •
22
MATERIALS AND METHODS
Host Response to PKX
This experiment was initiated on April I r 1987 and
terminated twenty weeks later on August 18, 1987, at the
Fish Disease Laboratory, University of California, Davis,
California.
Animals
Unexposed, age 0+ rainbow trout (10 g) from Hot Creek
California State Fish Hatchery were maintained at the Fish
Disease Laboratory UC Davis, and used as test animals.
Fish were held in 133 liter tanks supplied with 15 C well
water in a flow through system, and fed a commercial fish
feed at a daily rate of 1.5% body weight.
Fish were given prophylactic treatments of
nitrofurazone to prevent infections with Flexibacter
go
lumnaris, which is carried by the fish.
There was no
apparent mortality due to columnaris in either group of
fish during the study.
However, eleven weeks after
initiation of the study, the external parasite Costia
(Ichthyobodo sp.) caused a substantial loss (approximately
20%) in the PKD-infected group.
Fish were; subsequently
23
treated with formalin at a rate of 100 ppm. The control
group did not experience problems with Costia.
Experimental Protocol
PKX was obtained from kidneys of naturally infected
rainbow trout from the Hot Creek Hatchery.
Infection was
confirmed by wet mount followed by histologic examination.
Kidney tissue was removed, minced with an equal volume of
minimal essential media (MEM), forced through a sieve and
mixed 1:1 with phosphate buffered saline.
Eighty unexposed
rainbow trout were anesthetized with MS-222 (.04mg/ml) and
received 0.2 ml PKD-infected kidney homogenate via an
intraperitoneaI injection just anterior to the pelvic fins.
The dose of PKX per fish as determined by examination of
ten fields at 40x was 1.45 x 103 parasites.
Sixty control
fish were each injected with 0.2 ml MEM.
Tissue Collection and Processing
At three, five, seven, ten and twenty weeks
post-injection (PI) ten PKD-inoculated trout were randomly
collected.
Ten control fish were sampled at six, ten and
twenty weeks PI.
Fish were killed with an overdose of
MS-222 and kidney tissue was removed immediately and fixed
for light and electron microscopy.
\
24
For light microscopy, tissue was collected from
posterior kidney, preserved in Davidson’s fixative for 24
hours, transferred to 70% ethanol, embedded in paraffin and
sectioned at five micrometers.
Sections were stained with
H&E, Giemsa, hematoxyIin-PAS, Masson's trichrome, and
Turnbull's blue.
Corresponding kidney tissue was fixed in 2.5%
glutaraldehyde for 18 hours at 4 C , buffered to pH 7.2 in
.IM cacodylate phosphate buffer; then post-fixed in 1%
aqueous osmium tetroxide, dehydrated in graded ethanols,
embedded in Spurr's embedding medium, and sectioned on a
Sorvall MT 5000 ultramicrotome.
Thick sections were
stained with toluidine blue to select representative areas
and PKX parasites.
Thin sections (45 nm thick) were
stained with uranyl acetate and Reynold's lead citrate and
examined by transmission electron microscopy (TEM) with a
JEOL-IOOCX electron microscope.
Tissue collection and fixation for all sampling
periods except week seven were dorte by Fish Disease
Laboratory personnel at UC Davis, and sent to the Fish
Technology Center, Bozeman, Montana.
I traveled to UC
Davis to confer with the staff and personally collect the
week seven PI samples.
25
Host Response to Clay
This experiment was conducted at the U.S.
Fish and
Wildlife Service, Fish Technology Center, Bozeman, Montana.
The study was initiated on September 2, 1987 and terminated
20 weeks later on January 14, 1988.
Animals
Disease free age 1+ rainbow trout (190 g) were
maintained similarly at the Bozeman Fish Technology Center
but at a water temperature of 10 C. These fish were used as
comparative controls to observe the cells involved in a
chronic inflammatory response to a non-infectious
substance.
Experimental Protocol
Preliminary data using finely ground bentonite clay as
an inoculum demonstrated a granulomatous response.
Therefore, bentonite clay was used as the test material in
the comparative controls.
The clay was mixed with sterile
0.85% NaCl (6g clay/40ml saline) and 0.8ml injected IP into
each of eighteen anesthetized fish.
Six control fish were
each injected IP with 0.8ml sterile saline.
Previous
experiments injecting the inoculum at the same rate of PKD
kidney homogenate (0.02 ml/g body weight) produced high
26
mortality.
Studies indicated that bentonite clay
administered by IP injection at the rate of 0.004 ml/g body
weight was sufficient to induce a granulomatous response.
Tissue Collection and Processing
Sampling began one week PI because studies with
colloidal carbon have shown carbon containing macrophages
in kidney at four days PI (Ellis and Munroe, 1976).
Three
comparative controls were sampled at one, three, five,
seven, ten and twenty weeks post-injection.
Half the
saline controls were collected at five weeks and the
remainder at the termination of the study.
Posterior
kidney was dissected and processed in the same manner as
mentioned above for light and electron microscopic
examination of PKD-infected kidney tissue.
Pyloric caeca
from these fish were also dissected, processed and examined
because of visceral granulomata formed in response to clay.
Cell Classification
Kidney interstitial cells that responded to inoculum
were classified as; lymphocytes, polymorphonuclear
leukocytes (PMN), macrophages, thrombocytes and hemopoietic
blast cells.
Cells were classified at the light
microscopic level using the following criteria based on the
27
■literature (Ellis, 1976; Cannon et al., 1980; Yasutake and
Wales, 1983; Hightower et al., 1984) for hematoxyIin-eosin
stained sections:
Lymphocyte - small spherical cells, eccentrically
located spherical, reddish-purple nucleus with a narrow
band of lightly basophilic cytoplasm that frequently
contained granules.
PMN - round to ovoid cell with bilobed to
multilobulated, eccentric nucleus and blue-gray cytoplasm.
Macrophage - large, reniform, reddish-purple nucleus
with moderately basophilic cytoplasm that often contained
fine granules and vacuoles.
Melanomacrophage - macrophage containing
brownish-black melanin granules.
Thrombocyte - the long form has an elliptical, dense
basophilic nucleus often with one or two clefts, very light
basophilic cytoplasm that can appear streaked.
The
spheroid form is indistinguishable from small lymphocytes.
Plasma cell - eccentrically located nucleus,
reddish-purple with eosinophilic cytoplasm.
Blast cell - large pale, centrally located nucleus
with prominent blue-purple nucleoli and basophilic
cytoplasm.
The ultrastructural criteria primarily for plaice
(Ferguson, 1976), carp (BlaxhalI, 1983; Cenini, 1984) and
28
channel catfish, Ictalurus piinctatus, (Cannon et al., 1980)
leukocytes was used as a basis for cellular classification
and are as follows:
Lymphocyte - 3-10 urn, large sometimes indented nucleus
with dense compact chromatin, thin rim of cytoplasm with
limited rough endoplasmic reticulum, few elongate
mitochondria and small pseudopodia.
PMN - 8-10 urn, irregular and sometimes eccentric
nucleus with dense, patchy chromatin, no pseudopodia and
specific granules (crystalline or fibrillar) in the
cytoplasm.
Macrophage - 12-20 urn, eccentric nucleus with loosely
packed chromatin, well developed rough endoplasmic
reticulum, prominent Golgi apparatus, vesicles of varying
size and electron-density, few to numerous ovoid
mitochondria, pseudopodia.
The cytoplasm often contains
small, round, dark granules with an electron-lucent rim and
phagolysosomes.
Melanomacrophage - smilar to macrophage above, but
contains a large number of membrane bound vesicles and
eleptron-dense melanin granules.
Thrombocyte - 5-8 um, indented nucleus with cross
hatched heterochromatin and euchromatin, few mitochondria,
sparse rough endoplasmic reticulum, small Golgi apparatus,
prominent electron-lucent vesicles, centrioles and numerous
29
microtubules.
Plasma cell - 10-12 urn, eccentric nucleus with
radially arranged chromatin and distinct nucleolus, and
abundant rough endoplasmic reticulum.
Blast cell - large, oval, euchromatic, centrally
located nucleus and prominent nucleolus.
Cytoplasm
contains few mitochondria and numerous ribosomes.
30
RESULTS
The results presented here are based on
interpretations following the examination of paraffin
sections and electron micrographs.
The severity of lesions
associated with PKD and the prevalence of PKX varied from
moderate to extensive in kidney tissue collected at each
sampling period.
PKX was not found in control samples.
PKX
Typical PKX was detected in the kidney vasculature,
interstitium, tubular epithelium and lumina of infected
fish.
By light microscopy, the primary cell was lightly
eosinophilic with a prominent, deeply staining nucleolus
and PAS positive cytoplasmic granules.
Ultrastructurally,
primary cells were uninucleate with a euchromatic nucleus
and prominent electron-dense nucleolus, electron-dense
plasmalemma, granular cytoplasm and characteristic
haplosporosome-like bodies.
These "hapIosporosomes" had an
electron-dense matrix with electron-lucent bars one-half to
one-third the diameter, especially prominent in those near
the plasmalemma.
The cytoplasm contained abundant rough
31
endoplasmic reticulum, spherical to elongate mitochondria
with plate-like cristae, lipoid and multivesicular bodies.
'
The lipoid and multivesicular bodies appeared to be more
prominent in parasites that contained daughter cells.
Vegetative reproduction of interstitial. PKX by binary
fission and internal cleavage was observed.
Primary cells
containing up to four daughter cells were seen in infected
kidneys.
Daughter cells were encircled by the outer cell
membrane and contained prominent, electron-dense nucleoli,
few mitochondria, little rough endoplasmic reticulum,
abundant free ribosomes and occasionally lipoid bodies
(Figure I).
"Haplosporosomes" were never seen in daughter
cells but were found adjacent to the primary cell membrane
surrounding the daughter cell.
The secondary cell often
appeared to be separating from the enveloping primary cell
(Figure 2).
PKX was observed in kidney tubular epithelium five
weeks PI and tubular lumina ten weeks PI.
Intraluminal
forms varying from small, uninucleate to larger,
multinucleate, eosinophilic cells were observed in
paraffin sections.
Small parasites that resembled daughter
cells and multinucleate sporoblasts were found in distal
tubular lumina by electron microscopy.
Multilaminate
bodies were seen in the enveloping cell of the intraluminal
sporoblast, but polar capsules were not observed.
32
■ ■ ■ ■ ■ ■
Figure I.
PKX containing internal daughter cell (Dc);
haplosporosomes (arrowheads), nucleus (Nu)f
lipoid body (arrow), mitochondria (Mi)(x9000).
33
Figure 2.
PKX containing daughter cells (*) that appear to
be separating (arrow) from enveloping primary cell;
haplosporosomes (arrowheads) (x7800).
34
Host Response to PKX
Week Three
PKX were observed infrequently by light microscopy in
five of ten kidneys collected three weeks PI.
They were
observed in the hemopoietic tissue, sinusoids and
peritubular capillaries; most appeared to have macrophages
closely adhered to the cell surface of the parasite (Figure
3a).
Several parasites were found free in the circulation.
Several stages of development were observed; small
condensed eosinophilic cells resembling daughter cells,
primary cells and primary cells containing daughter cells
(Figure 3b).
In one sample, small cells, possibly
degenerate PKX, were observed in the tubular epithelium and
lumina (Figure 4a).
In several samples, focal areas of hemopoietic tissue
appeared intensely basophilic, apparently due to increased
cellularity.
Numerous mitotic figures were evident in
these areas.
Melanomacrophages were found scattered
throughout the hemopoietic tissue, except for one sample in
which there was a marked increase in numbers and
concentration.
35
Figure 3.
a) PKX (arrowhead) in sinusoid with macrophage
(arrow) closely adhered to cell surface, three
weeks PI (x400). b) PKX (arrowhead) containing
daughter cell in kidney interstitium; macrophage
(arrow) (x400).
36
Figure 4.
a) Vacuoles (arrows) containing cellular
material in tubular epithelium (x400).
b) Lymphocytes (arrows) migrating through
tubular epithelium (xl60).
37
Numerous lymphocytes were seen migrating through
tubular epithelium (Figure 4b).
The major tubular change
observed three weeks PI was vacuolation of proximal tubular
epithelium with hyaline droplet degeneration and
accumulations of an eosinophilic granular material in most
tubular lumina.
Examination by electron microscopy of kidneys
collected at three weeks PI confirmed,findings at the light
✓
microscopic level, i.e. large cytoplasmic droplets,
vacuolation of tubular epithelium, migrating lymphocytes,
mitoses of hemopoietic cells and finely granular material
in tubular lumina (Figure 5).
However, the presence of any
developmental stages of PKX were not confirmed by
ultrastructural examination of tissue samples.
Endothelial cells lining kidney sinusoids and
peritubular capillaries appeared activated as evidenced by
the abundance of pinocytotic vesicles adjacent to the
luminal plasmalemma and cytoplasmic protrusions.
Numerous
lymphocytes were seen in the peritubular capillaries.
Circulating macrophages were observed attached to the inner
surface of vessel walls (Figure 6).
Macrophages were seen
migrating through tubular basal lamina and into tubular
epithelium.
Occasionally, macrophages containing
Figure 5.
Migrating lymphocyte (Ly) and vacuoles (Va) in
tubular epithelium and granular material (Gr) in
tubular lumina, three weeks PI (x4680).
39
Figure 6.
Circulating macrophages (Ma) attached to
endothelium (En) of vessel wall, three weeks PI
(x5200).
.40
degenerate cellular material in phagocytic vacuoles were
overlying endothelial cells.
Large, electron-dense, nucleolar-like material, was
seen in macrophages in several tissue samples collected at
three weeks PI.
An unidentifiable cell observed with
relative frequency in the kidney interstitium (Figure 7) at
three weeks PI was not found in subsequent samples or
controls.
The cell was small, contained electron-dense
cytoplasm, numerous vacuoles, few mitochondria, numerous
long pseudopodia and often a deeply indented nucleus with
surrounding microtubules (Figure 8).
Disruption of
nuclear membranes, swollen mitochondria or condensation of
organelles suggestive of degeneration was not observed in
these cells.
Week Five
At five weeks PI interstitial inflammation was evident
in kidney samples from all ten fish.
PKX was identified in
seven samples; one heavily, one moderately and the rest
lightly infected.
Samples collected at this time exhibited
a wide range of severity of lesions, from small localized
areas of hemopoietic proliferation, vacuolation of proximal
tubular epithelium and no identifiable PKX (Figure 9a) to
advanced lesions with massive hemopoietic proliferation.
41
Figure 7.
Unidentified cell (Uc) found in kidney
interstitium, three weeks PI (x5200).
42
Figure 8.
Unidentified cell with electron-dense cytoplasm,
vacuoles, indented nucleus (Nu) and pseudopodia
(arrowhead) (xl5,000) .
43
Figure 9.
a) Mild PKD (xl60). b) Severe PKD, five weeks
PI; PKX (arrowheads) (xl60).
44
few intact tubules and numerous PKX cells (Figures 9b).
Although inflammation was evident in all ten fish, PKX was
not identified in three samples that exhibited mild
hemopoietic proliferation.
Prominent features observed by light microscopy were
intense basophilia of hemopoietic tissue, numerous mitotic
figures, and displacement of renal elements by increased
hemopoietic cells.
There was a marked decrease in tubular
density in two samples.
Cells infiltrating the
interstitium consisted largely of lymphocytes and
macrophages.
One small granulomatous foci with a single PKX located
in the center Was seen in the hemopoietic tissue of one
kidney sample.
PKX was observed in tubular epithelium of
only one fish that was heavily infected with PKX and
exhibited advanced lesions (Figure 10).
Most PKX were
surrounded by macrophages and occasionally attached to
vessel walls.
Severe vasculitis with areas of occlusion was seen in
the renal vein of one sample (Figure 11a).
Macrophages
appeared to be adhering to the surface of PKX cells that
were free within the vessel, on the edge of and within the
lesion.
The lesion was comprised of parasites,
macrophages, lymphocytes, and erythrocytes (Figure 11b).
45
Figure 10.
a) PKX in tubular epithelium (arrow) and lumina
(* ) (x400). b) PKX in tubular epithelium
(arrow) and hemopoietic tissue (arrowhead),
five weeks PI (x400).
46
Figure 11.
a) Vascular lesion in renal vein, five weeks
PI; PKX (arrowhead), lymphocytes (*) (xl60).
b ) Vascular lesion containing PKX (arrowheads),
lymphocytes (*), macrophages (arrows) and
erythrocytes (double arrow) (x400).
47
The endothelium and muscularis were often disrupted where
inflammatory lesion was attached, possibly allowing
macrophages, lymphocytes and PKX to migrate through the
vessel wall into the hemopoietic tissue.
A brownish-green
pigment which stained negative for hemosiderin, was
observed scattered throughout the renal vein, hemopoietic
tissue and occasionally lining tubular lumina.
Numerous lymphocytes were seen migrating through the
epithelium of collecting tubules, however,. this was also
observed in controls and may represent a normal condition.
A marked increase in melanomacrdphages was noted in five
weeks PI in experimentally-infected fish and among samples
from the control group at six weeks.
Apparently this was
in response to the infectious agent, E\
columnaris,
present in both groups of fish.
Electron microscopy showed that PKX found in the
interstitium had at least one macrophage, usually two or
more closely adhered to their surface (Figure 12).
Lymphocytes were often seen in close proximity but not
directly adjacent to the parasite.
PKX observed in
the circulation appeared to be in the process of being
engulfed by macrophages (Figure 13).
Macrophages
containing degenerate cellular components, possibly PKX
were observed in tubular epithelium of several tubules
48
Figure 12.
Macrophages (Ma) closely adhered to the cell
surface of PKX; lymphocytes (Ly), nucleus (Nu)
(x5000).
49
Figure 13.
PKX in kidney circulation partially (double
arrows) and completely (arrow) engulfed by
macrophages; daughter cell (Dc), nucleus (Nu)
(x5000).
50
(Figure 14).
In contrast to three week samples,
peritubular capillary lumina appeared collapsed from
increased cellularity or engorged with blood cells.
■
I
Week Seven
Eight of the ten fish sampled at seven weeks PI had
grossly swollen kidneys.
Histologically, PKX was found in
all ten kidney samples; four were heavily, three moderately
and three lightly infected with the parasite.
There was a
marked decrease in tubular density in all ten samples.
Intact as well as degenerate PKX, often with intact
daughter cells, were observed in kidney tissue.
The intact
daughter cells appeared as small condensed eosinophilic
cells, similar to the early stage of PKX seen at three
weeks PI.
Cells, predominantly macrophages, were found
adhered to all the parasites observed at this time.
The characteristic feature of samples taken at seven
weeks PI was an epithelioid-like granulomatous response to
PKX. Granulomatous lesions replaced hemopoietic tissue to
varying degrees throughout the kidney interstitium.
PKX
was usually found in the center of a "whorl" of macrophages
(Figure 15).
Epithelioid-like macrophages were the
51
Figure 14.
Macrophage (Ma) containing degenerate cellular
material (arrowhead), possibly PKX, in tubular
epithelium (*), five weeks PI (x5200).
52
Figure 15.
a) Granulomatous lesions seven weeks PI; PKX
(arrowhead) (xl60). b ) PKX (arrowhead) in the
center of a whorl of macrophages (arrows) (x400).
53
predominant cells found in week seven samples.
Fibroblasts
were found only widely scattered throughout the
inflammatory tissue.
The most interesting feature observed at the
X
ultrastructural level was the abundance of plasma cells,
found only occasionally at previous sample times.
Plasma
cells were not found attached to or in close proximity to
parasites but were abundant throughout the kidney
interstitium.
Epithelioid cells were the predominant cell
type found in kidney samples (Figure 16).
Epithelioid
cells were morphologically similar to macrophages but they
were usually larger, elongate and did not contain
phagolysosomes.
The lack of collagen fibrils in the
extracellular space close to the cell distinguished these
cells from fibroblasts.
Macrophages were seen migrating
through the tubular basal lamina and into tubular
epithelium.
Peritubular and sinusoidal spaces were often
collapsed.
Tubular basal lamina appeared thickened.
Autophagic vacuoles, myelin figures and clumped chromatin,
all of which are indicative of degeneration, were often
noted in tubular epithelium.
Figure 16.
Epithelioid tissue seven weeks PI (x5200).
\
X
55
Week 10
All ten fish sampled at ten weeks PI were positive for
PKX. Six fish were heavily, three moderately, and one
lightly infected.
Intraluminal forms of PKX were observed
in kidneys from three fish.
Macrophages were attached to
most of the parasites in the kidney interstitium (Figure
17a) and blood vessels, but host cells were not found
associated with the intraluminal forms of PKX (Figure 17b).
PKX cells that had macrophages adhered to their surface
appeared to be undergoing degeneration, but daughter cells,
of which there were often two or more, were usually
intact.
Epithelioid tissue was markedly reduced in ten weeks
PI samples as compared to seven weeks PI samples.
Proliferative hemopoietic tissue was prominent with diffuse
epithelioid tissue scattered throughout the interstitium.
PKX cells were mostly found within epithelioid tissue.
intact tubular elements remained at this time.
Few
Numerous
lymphocytes were seen in remaining tubules.
An interesting feature of kidney tissue from ten weeks
PI was the presence of brownish-black, melanin-like
granules, mostly lining tubular lumina (Figure 17b).
56
Figure 17.
a) Macrophage (arrow) adhered to surface of PKX
(arrowhead) in kidney interstitium;
intraluminal PKX (*) (x400). b ) MeIanin-Iike
material (arrow) lining tubule lumina ten weeks
PI; intraluminal PKX (*) (x400).
57
Staining for hemosiderin was negative and identification of
this material by ultrastructural examination was
unsuccessful.
Most commonly, PKX was seen by electron microscopy
with at least one macrophage, usually two or more,
adhered to the surface completely surrounding the parasite
(Figure 18).
Macrophages containing electron-dense
granules with an electron-lucent halo, presumably
lysosomes, were often observed in close proximity to the
plasmalemma bordering PKX (Figure 19).
Lymphocytes were
often, but not always found in close association with the
outermost macrophage plasmalemma but rarely adjacent to
PKX (Figure 20).
At ten weeks PI, the hemopoietic tissue was comprised
primarily of leukocytes; predominantly macrophages,
immature and mature lymphocytes.
Few erythrocytic
precursors were observed in the hemopoietic tissue.
In
comparing tissue samples to week three, hypercellularity
was evident by the lack of intercellular spaces and the
compressed appearance of peritubular capillaries and
sinusoids.
58
mm
Figure 18.
•
m•
Macrophages (Ma) closely adhered to the surface
of PKX; daughter cell (Dc) (x5720).
59
Figure 19.
Electron-dense granules (arrows) in close
proximity to plasmalemma adjacent to PKX;
haplosporosomes (arrowheads), daughter cell
(Dc), nucleus (Nu) (xlO ,000).
60
Figure 20.
PKX with macrophages (Ma) closely adhered to
cell surface and lymphocyte (Ly) closely
associated with macrophages; daughter cell
(Dc), nucleus (Nu) (x4200).
61
Macrophages:- were again, found migrating through the basal
lamina and into distal tubular epithelium (Figure 21).
Tubular epithelium often showed signs of degeneration, such
as swollen mitochondria, myelin figures, cytoplasmic
vacuolation, and a thickened basal lamina, fibroblasts and
epithelioid cells.
Like plasma cells seen in week seven,
thrombocytes were more numerous in these tissue samples
than previously observed (Figure 22).
Week Twenty
The study was terminated at twenty weeks PI.
PKX was
not identified in the kidney interstitium or tubular lumina
in any of these tissue samples.
Small granulomatous foci,
vacuolated tubules and regenerating tubules were the most
prominent features observed by light microscopy.
AlI
kidneys appeared to be recovered or recovering from PKD as
evidenced by the reduction in hemopoietic and granulomatous
tissue, increase in renal elements, and numerous
regenerating tubules (Figure 23).
Cells in regenerating
tubules were distinguished by their deeply basophilic
cytoplasm.
62
Figure 21.
Macrophage (Ma) migrating through basal lamina
(arrow) into tubular epithelium, ten weeks PI;
PKX (x4200).
63
Figure 22.
Thrombocytes (Th) found at ten weeks PI
(x5460).
64
Figure 23.
Regenerating tubule (arrow), twenty weeks PI
(x400).
65
Thickened basal lamina, macrophages in tubules,
regenerating tubular epithelium and scattered fibroblasts
were the most common ultrastructural features of these
kidney samples (Figure 24)'.
Cells of regenerating tubular
epithelium were squamous to cuboidal in shape, had
large nuclei with diffuse chromatin, prominent nucleoli and
few or no microvilli.
Host Response to Clay
During the twenty week experiment, there was no
mortality of fish injected with bentonite clay and saline.
The control fish injected only with saline did not show any
changes in the pyloric caeca or kidney tissue samples.
Week One
Histologically, clay particles were visible in tissue
surrounding pyloric caeca.
Macrophages appeared to be the
only cell type responding to the presence of clay in the
visceral cavity.
The most characteristic feature in the
kidney was the increase in melanomacrophages found in the
66
Figure 24.
Thickened basal lamina (arrow) and regenerating
tubular epithelium (Tu), twenty weeks PI
(x4000).
67
interstitium.
Some melanomacrophages contained clay
• -ri
particles as evidenced by the blue color of clay in
sections stained with Giemsa.
Most proximal tubules
appeared heavily vacuolated.
Electron microscopic examination of the kidney samples
revealed the presence of clay particles in
melanomacrojphages and macrophages that appeared to be
overlying endothelial cells (Figure 25).
Endothelial
cells, especially those lining the peritubular capillaries
were swollen and markedly vacuolated.
In contrast to fish
with PKD, PMNs were observed more frequently in the
hemopoietic tissue.
/
Week Three
Grossly, visceral adhesions were first noted in fish
collected at week three.
An inflammatory response to the
X
clay was evident in tissues surrounding pyloric caeca
samples.
Bentonite clay induced distinct visceral
.granulomas that were scattered throughout the fatty tissue.
The granulomas were mainly comprised of macrophages
containing clay particles and a few multinucleate giantcells.
68
MPHHVI
v-
<
-...*■>-;'■•.«-A1*/
.
.... f.
'
,
A J'J. M
,. .../.
•
■;
:.M
/
Figure 25.
Clay particle (arrow) contained in macrophage
(Ma) which is overlying endothelial cell (En);
tubular epithelium (Tu), mitochondria (Mi)
(xll,500).
69
Melanomacrophage proliferation in hemopoietic tissue
was more intense in the three weeks PI than one week PI
samples.
Again, sections stained with Giemsa revealed the
presence of small clay particles in these cells.
Clay
stained intensely blue with Giemsa.
Ultrastructurally, activated endothelial cells were
observed with clay particles in their cytoplasm.
Clay
particles were also found again in macrophages and
melanomacrophages.
Tubular lumina, usually distal
segments, were almost occluded with a finely granular
material similar to that seen in PKD-infected fish at three
weeks PI.
Many large, electron-dense droplets, similar to
those seen in hyaline droplet degeneration, were found in
proximal tubular epithelium.
Week Five
Visceral granulomas were not seen in any of the
samples collected five weeks PI.
However, kidney tissue
from two of three fish sampled revealed an intense
proliferation of melanomacrophages in the hemopoietic
tissue
70
Melanomacrophages were the predominant cell type found
in kidney interstitium by electron microscopy (Figure 26).
However, clay particles had been phagocytosed mostly by
macrophages.
Week Seven
Massive visceral granulomas containing clay particles
of various sizes were observed at seven weeks PI.
Macrophages, epithelioid cells, and foreign-body giant
cells were the predominant cell types found in the lesion
(Figure 27a).
A lymphocytic infiltrate was not seen in
these granulomas.
Marked hypercellularity and a decrease
in tubular density in the hemopoietic tissue was noted in
kidney samples.
Melanomacrophages almost filled the entire
kidney interstitium (Figure 27b).
Clay particles were
difficult to discern in the&e areas of the concentration of
darkly staining melanomacrophages.
71
Figure 26.
Melanomacrophages (Mm) found in kidney
interstitium, five weeks PI (x5200).
72
Figure 27.
a) Visceral granuloma seven weeks PI; giant cell
(Gi) (xl60). b ) Melanomacrophages dominate the
kidney interstitium seven weeks PI (x400).
I
73
Electron microscopic examination of kidney tissue
showed clay particles mostly in macrophages (Figure 28)>
occasionally endothelial cells, and very seldom in
melanomacrophages although this was again the predominant
cell type.
An accumulation of large, electron-dense
absorption granules was noted in proximal tubular
epithelium (Figure 29).
Week 10
Visceral granulomas ranged from small, well
circumscribed lesions scattered throughout adipose tissue
to massive lesions invading the intestinal muscularis and
obliterating most of the fatty tissue.
Necrosis was noted
in several areas within large granulomas.
Lymphocytes
and numerous long spindle-shaped fibroblasts were seen in
the large, more advanced lesions (Figure 30a).
A moderate decrease in tubular density and
hypercellularity in the kidney interstitium were the
characteristic features of kidney tissue sampled.
Melanomacrophages and mononuclear cells were the
74
Figure 28.
Macrophage (Ma) with phagocytosed clay
particles (arrows) seven weeks PI (xlO,000)
75
Figure 29.
Large, electron-dense absorption granules
(Gr) in proximal tubular epithelium seven weeks
PI; nucleus (Nu) (x5460).
76
predominant cell types found in the interstitium.
Proximal
tubular epithelium appeared to have extremely vacuolated
cytoplasm and hyaline droplet degeneration was noted in
many of these tubules.
Cellular debris was evident in
lumina of several collecting tubules.
Regenerating tubules
were observed throughout the kidney tissue.
Melanomacrophages were seen in the kidney circulation
(Figure 30b).
In sections stained with Giemsa, clay
particles were difficult to distinguish in areas of
hemopoietic tissue containing large concentrations of
melanomacrophages, but easily identifiable in macrophages
not located near these areas.
Ultrastructural examination of kidneys revealed
results similar to seven weeks PI.
Plasma cells were first
noted in these samples, often adjacent to kidney tubules
(Figure 31).
Small, atypical barbell shaped mitochondria
suggestive of sublethal injury were often seen in tubular
epithelium (Figure 31).
77
Figure 30.
a) Lymphocytes (double arrow) and fibroblasts
(arrows) in visceral granuloma (x!60). b)
Melanomacrophages (arrowheads) in the kidney
circulation ten weeks PI (xl60).
78
Figure 31.
Plasma cell (Pl) adjacent to kidney tubular
epithelium with small barbelI-shaped
mitochondria (small arrows) ten weeks PI; basal
lamina (large arrow), erythrocyte (Er)
(x7600).
79
Week 20
Grossly, samples collected at the termination of the
study showed visceral adhesions along the intestine and
black streaks in the visceral fat and pyloric caeca.
Histologically, small to large granulomas, were found
scattered throughout the visceral fat, attached to and
invading spleen capsules and intestinal muscularis.
The
characteristic feature of these samples was numerous
melanocytes, not seen previously, found primarily in the
center of the granulomas (Figure 32).
Kidney tissue more closely resembled saline injected
controls at this time.
Tubular density appeared normal.
Hypercellularity, extremely vacuolated tubular epithelium,
and protein material in tubular lumina were markedly
reduced. Melanomacrophages were found primarily around
blood vessels and lightly scattered throughout the
interstitium.
Blast cells were evident and clay particles
were difficult to find in the hemopoietic tissue.
Electron
microscopic examination of samples confirmed the above
results.
80
Figure 32.
Melanocytes (arrows) in visceral granuloma
twenty weeks PI (xl60).
81
DISCUSSION
Developmental stages of PKX previously described by
Kent (1986), Rafferty (1986) and Clifton-Hadley et al.
(1987) were observed in this study.
The small condensed
eosinophilic form of PKX at three weeks PI and migration of
the parasite through tubular epithelium reported by Kent
(1986) were observed by light microscopy but not confirmed
by electron microscopy.
Experimental fish infected with
PKD appeared to mount an effective local cellular response
against PKX, interrupting maturation of the parasite.
Typical myxosporeans, have primary cells that eventually
disintegrate and release daughter cells, which may begin
the cycle again, go into the sporogenic phase or be
destroyed by the host tissue reaction (Lorn, 1987).
Intraluminal sporogenic forms were observed infrequently,
and were not found in any fish collected twenty weeks PI.
Complete sporulation did not take place as evidenced by the
lack of polar capsule formation.
Vegetative reproduction by binary fission of PKX
internal cells (Kent, 1986) and internal cleavage (Seagrave
et al., 1980) typical of the phylum Myxozoa (Lom et al.,
1983) was observed in this study.
The secondary cell or
generative cell has characteristic bundles of microtubules
I
82
close to the nucleus, numerous free ribosomes in the
cytoplasm, pseudopodia-like extensions and lack a
centriole.
It is this type of cell that is produced by the
released sporoplasm and is the initial stage of the
extrasporogenic cycle (Lorn, 1987).
Unidentified cells seen
early in the infection could represent the infective stage
of PKX, but may alternately represent foreign fish cells
from the kidney homogenate inoculum,
PKX provoked a severe host reaction characterized by
interstitial hyperplasia and granulomatous interstitial
nephritis.
This is in contrast to most myxosporeans which
invoke practically no tissue reaction (Lorn, 1987).
The
host inflammatory response seemed to inhibit both migration
of PKX to the tubular lumina and subsequent sporulation.
Infection rate and intensity of disease did not appear to
be dose related.
Prevalence of PKX and degree of
associated inflammation are dependent on the
immunocompetence of the host which is affected by several
factors, especially in parasitic infections (Woo, 1987).
Interstitial hyperplasia characteristic of PKD (Kent
and Hedrick, 1986; Clifton-Hadley et al., 1987) Was
observed in fish injected with PKX beginning three weeks PI
and extending through ten weeks PI.
This response can be
compared to bone marrow hyperplasia seen in many infectious
diseases of man.
Fish sampled at five weeks PI exhibited a
83
marked cellular response and by seven weeks PI chronic
inflammation was evident.
The increase in plasma cells
observed seven weeks PI was indicative of a humoral
response to PKX. Replacement of epithelioid tissue with
normal hemopoietic tissue at ten weeks PI may be indicative
of recovery not inflammation, since PKX was found mostly in
granulomatous lesions.
Thrombocytes, believed to be the
equivalent of mammalian platelets are probably part of the
healing process (Ellis 1981).
Fibrosis, typical of
resolution of an inflammatory lesion, was seen only to a
minor degree during later stages of the disease.
The sequential pathologic changes of PKD observed in
this study were similar to that reported by Clifton-Hadley
(1987).
However, fusiform hemosiderin crystals associated
with intravascular lesions and nodules of chronic
inflammation were not observed during the study.
Glomerular sclerosis as described by Ferguson and Needham
(1978) was not observed to any degree during the study.
Rafferty (1986) reported marked melanomacrophage
proliferation during early stages of experimentally induced
PKD and suggested melanomacrophages were involved in
processing PKX antigens.to stimulate the immune response.
This phenomenon was not observed during this study.
Clifton-Hadley (1987) found granules of melanin throughout
reactive tissue but few melanomacrophages, similar to the
84
findings in this study.
Melanin is a pigment known for its
ability to absorb free radicals and cations and render them
inactive.
However, melanin also has bactericidal
properties and has been associated with lipid peroxidative
tissue damage (Edelstein, 1971; Roberts, 1975).
Possibly,
melanin is mobilized to neutralize free radical and cation
activity and explains why the pigment has been commonly
observed in sites of infection (Agius, 1985).
Evidence from this study indicates that macrophages
are the predominant host cell type associated with PKX
throughout the disease.
This is not surprising since the
macrophage is the most important cell type in host defense
against invasion by infectious or noninfectious foreign
material (Corbel, 1975).
Fish macrophages remove debris,
may destroy intracellular microorganisms, may uptake and
process antigen, may kill antigen deviant cells, and may
actively secrete various substances (Agius, 1985).
Little
is known about the factors that affect the phagocytic
response of macrophages in fish.
It has been suggested
that their role is more primitive and restricted than that
seen in mammals (Ellis, 1981).
In this study, PKX was not
eliminated until twenty weeks PI even though macrophages
were found closely adhered to the cell surface of most
parasites at five weeks PI.
PKX may delay destruction by
prolonging the killing mechanisms of the macrophages,
85
possibly by producing a substance that prevents fusion of
lysosmes or inhibits the production of a sufficient number
of lysosomes.
Melanomacrophages were the predominant cell type found
in kidney samples from fish injected with clay.
However,
the clay particles that gained access to the circulation
were phagocytosed primarily by macrophages in the
hemopoietic tissue in kidney.
Perhaps these macrophages
later became melanomacrophages.
understood in melanomacrophages.
Melanogenesis is poorly
It is not known if
melanin is phagocytosed or manufactured in these cells.
Normally, melanomacrophages accumulate steadily with
age and kidneys of older fish may contain large numbers,
but this process is often accelerated in the course of some
diseases (Agius, 1985).
Melanonmacrophages contain melanin
that is different than that found in melanocytes of the
dermis and has been associated with various pathological
conditions (Roberts, 1975).
Melanomacrophage centers are
thought to represent primitive analogues of germinal
centers found in birds and man (Roberts, 1975).
All
teleosts, except salmOnids, have distinct melanomacrophage
centers which are considered to be an integral part of the
reticuloendothelial system (Roberts, 1975).
Inert
particles such as carbon are known to be phagocytosed by
macrophages that aggregate within melanomacrophage centers
86
(Agius, 1985).
It has been suggested that
melanomacrophages are sensitive indicators of stressful
conditions and are useful as health monitors (Wolke et al.,
1985).
The PMN response to infection is not as important in
fish as it is in mammals (Corbel 1975).
PMNs were
'
scattered throughout kidney interstitium during the study,
but never constituted a major portion of the lesion.
Many
authors (Sakai, 1984; Suzuki,1986) have reported that fish
neutrophils are phagocytic.
Clifton-Hadley (1987) found
what he believed to be neutrophils surrounding PKX. In this
study neutrophils were not phagocytic nor were they found
associated with PKX. The precise role of the neutrophil and
melanin pigment are poorly understood in fish diseases
(Ellis, 1981).
This study has demonstrated an effective cellular
response, dominated by macrophages, against infectious and
non-infectious foreign material.
The inflammatory response
in fish is considerably slower than that seen in higher
vertebrates, but regenerative capacity is high in fish
(Ellis, 1981).
Resolution of interstitial inflammation was
observed without fibrosis in both experimental groups by
twenty weeks PI.
Small localized areas of chronic
inflammation were still evident but tubular and hemopoietic
density were normal.
87
There are presently many gaps in understanding the
fish immune system (Woo, 1987).
There is general confusion
over the cellular components of the defense system in fish
because mammalian hematological terms have been applied
without evidence that functional similarities exist.
In
similarity to mammals there is production of specific
immunoglobulin, and phagocytic and cytotoxic cells.
Distinctly different, however, are reduced and structural
differences in immunoglobulin classes and the absence of
lymph nodes (Ellis 1981).
The teleost kidney executes
functions that are attributable to mammalian lymph nodes
and bone marrow.
Interspecies discrepancies of ultrastructural features
in leukocytes were encountered in this study.
Rainbow
trout cells most often resembled those described in channel
catfish.
However, neutrophils seen in this study had
segmented or multilobed nuclei.
Hemopoietic cell types in
fish are still far from being adequately charactertized,
particularly the immature forms.
Differences in
hemopoietic cells among species, especially those of
different water temperature regimes and environmental
conditions, need to be investigated.
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